Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg 2؉ -dependent cytotoxic event upon binding of the toxin to its receptor BT-R1. Here we show that binding of Cry toxin to BT-R1 provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G␣s) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase͞protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R1 in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides.Cry toxin ͉ protein kinase A ͉ cadherin receptor ͉ cAMP ͉ signal transduction

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.

The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxinreceptor interactions in cytotoxicity, we established a cellbased system utilizing High Five insect cells stably expressing BT-R 1 , the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R 1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R 1 , which apparently activates a Mg 2 þ -dependent cellular signaling pathway.

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Bacillus thuringiensis (Bt) is a unique bacterium in that it shares a common place with a number of chemical compounds which are used commercially to control insects important to agriculture and public health. Although other bacteria, including B. popilliae and B. sphaericus, are used as microbial insecticides, their spectrum of insecticidal activity is quite limited compared to Bt. Importantly, Bt is safe for humans and is the most widely used environmentally compatible biopesticide worldwide. Furthermore, insecticidal Bt genes have been incorporated into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture.This review highlights what the authors consider the most relevant issues and topics pertaining to the genomics and proteomics of Bt. At least one of the authors (L.A.B.) has spent most of his professional life studying different aspects of this bacterium with the goal in mind of determining the mechanism(s) by which it kills insects. The other authors have a much shorter experience with Bt but their intellect and personal insight have greatly enriched our understanding of what makes Bt distinctive in the microbial world. Obviously, there is personal interest and bias reflected in this article notwithstanding oversight of a number of published studies. This review contains some material not published elsewhere although several ideas and concepts were developed from a broad base of scientific literature up to 2010.

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined. Molecular & Cellular Proteomics 3:501-509, 2004.A human red blood cell (RBC) 1 is in residence in the human circulatory system for 120 days carrying oxygen from the lungs to all tissues within the body and carbon dioxide from the tissues back to the lungs. An RBC is an 8-m biconcave disk bounded by a plasma membrane. The major cytoplasmic constituent is hemoglobin, which is responsible for binding and releasing oxygen and carbon dioxide. On the cytoplasmic surface of the plasma membrane is a two-dimensional meshwork of proteins referred to as spectrin membrane skeleton. The spectrin membrane skeleton renders elasticity and flexibility to an RBC, allowing it to pass through vessels and capillaries that narrow to 1 m in diameter (1).Because of the ease in obtaining RBCs and because they lack internal organelles, the plasma membrane of this cell type has been studied extensively. The functions of hemoglobin are also well documented. Based on four decades of study, the identity, function, and topology of many RBC membrane proteins have been determined (1-3). With the advent of modern mass spectrometry (MS) and associated proteomic techniques, determination of the RBC proteome is now plausible. This kind of approach is a necessary first step in understanding how the RBC proteome becomes altered in various hematologic disorders. With this goal in mind, we utilized ion trap ...

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